Chemico-Biological Interactions 259 (2016) 122e132

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Chemico-Biological Interactions

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Design and synthesis of N-substituted-2-hydroxyiminoacetamides and interactions with

Nikola Marakovic a, Anamarija Knezevic b, Vladimir Vinkovic b, Zrinka Kovarik a, * Goran Sinko a, a Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10 000 Zagreb, Croatia b RuCer Boskovic Institute, Bijenicka cesta 54, HR-10 000 Zagreb, Croatia article info abstract

Article history: Within this study, we designed and synthesized four new oxime compounds of the N-substituted 2- Received 9 January 2016 hydroxyiminoacetamide structure and evaluated their interactions with (AChE) Received in revised form and (BChE). Our aim was to explore the possibility of extending the dual-binding 20 April 2016 mode of interaction between the enzyme and the inhibitor to a so-called triple-binding mode of inter- Accepted 25 May 2016 action through the introduction of an additional binding moiety. N-substituted 2- Available online 26 May 2016 hydroxyiminoacetamide 1 was prepared via BOP catalyzed amidation of hydroxyiminoacetic acid with 3-azido-1-phenylpropylamine. An azide group enabled us to prepare more elaborate structures 2e4 by Keywords: e Oxime antidotes the copper-catalyzed azide-alkyne cycloaddition. The new compounds 1 4 differed in their presumed Azide-alkyne cycloaddition AChE peripheral site binding moiety, which ranged from an azide group to functionalized heterocycles. Organophosphorus compounds Molecular docking studies revealed that all three binding moieties are involved in the non-covalent Inhibition interactions with ChEs for all of the four compounds, albeit not always in the complete accordance Selectivity with the proposed hypothesis. All of the four compounds reversibly inhibited the ChEs with their in- hibition potency increasing in the same order for both enzymes (1 < 2 < 4 < 3). A higher preference for binding to BChE (KI from 0.30 mmol/L to 130 mmol/L) over AChE (KI from 50 mmol/L to 1200 mmol/L) was observed for all of the compounds. Compounds were screened for reactivation of -, - and VX-inhibited AChE and BChE. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Gly122, Ala204), an acyl-binding pocket (Phe288, Phe290) and a binding site (Trp86, Tyr337, Phe338) [4,5]. The two sub- Acetylcholinesterase (AChE; EC 3.1.1.7) is a key enzyme for the sites serve as the recognition sites for the ligands that bind to the regulation of transmission in both the central and pe- AChE governing their mechanism of interaction. Depending on the ripheral nervous system that catalyzes the hydrolysis of the established interactions, ligands can be described as PAS-binding or neurotransmitter (ACh) [1]. The decline of hippo- the CAS-binding. Out of several anti-AD drugs (Fig. 1), galanth- campal and cortical levels of ACh is a characteristic of Alzheimer’s amine (half-maximal inhibitory concentration (IC50) of 2.01 mmol/L disease (AD), a disabling and fatal neurodegenerative disease in human AChE) [6,7] and (IC50 of 0.082 mmol/L in manifested by memory loss and learning deficits [2]. Thus, today’s mouse AChE) [8,9] bind to the CAS, while propidium iodide (IC50 of drugs designed for the treatment of AD are reversible AChE in- 1.1 mmol/L in mouse AChE) [10] binds in the PAS region [11]. In- hibitors that block the enzyme active site leading to an increase of hibitors that bind to PAS and CAS simultaneously include both ACh levels and, in turn, to the alleviation of disease symptoms [3]. symmetrical (e.g. bistacrine, Kd of 250 nmol/L in fetal bovine serum The active site of AChE is a 20 Å deep gorge divided into two sub- AChE) [12,13] and non-symmetrical analogues (e.g. syn- sites; the peripheral anionic site (PAS) (Tyr72, Tyr124, Trp286) TZ2PA6, Kd of 0.41 nmol/L in mouse AChE) [14,15],aswellas located at the entrance of the gorge, and the catalytic site (CAS) (Kd in hAChE of 3.35 nmol/L (R-donepezil), 17.5 nmol/L located close to the bottom of the gorge. CAS is composed of the (S-donepezil)) an anti-AD drug that binds with a basic nitrogen in catalytic triad (Ser203, His447, Glu334), an oxyanion hole (Gly121, the CAS and an aromatic system in the PAS [16,17]. Crystal struc- tures of AChE-inhibitor complexes have shown that inhibitors * Corresponding author. usually interact with gorge residues via areneearene (p-p) E-mail address: [email protected] (G. Sinko). http://dx.doi.org/10.1016/j.cbi.2016.05.035 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved. N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 123

Fig. 1. Structures of inhibitors, and reactivators of organophosphorus -inhibited cholinesterases. interactions. Trp86, which is essential for the interaction with the [8,17,23,24]. Several selective BChE inhibitors have also been trimethylammonium group of ACh, is considered crucial for the described, including bambuterol and ethopropazine (Fig. 1) [41e45]. stabilization of CAS-binding ligands through cation-p and p-p in- Using ChE active site differences for designing selective inhibitors teractions together with the Tyr337, and His447 of the catalytic could help develop improved AD drugs and reactivators of OP nerve triad [18e20]. On the other hand, PAS-binding ligands are stabilized agent-inhibited enzymes. through p-p interactions with Tyr72, Tyr124, and Trp286 [21e23]. In this study, we designed and synthesized four new compounds The acute toxicity of organophosphorus (OP) nerve agents (e.g. to probe the possibility of simultaneous non-covalent triple-bind- , , sarin, VX) is due to their irreversible inhibition of ing between the inhibitors and the ChE. Our results could lead to AChE by covalently binding to the catalytic serine residue which the discovery of more selective ChE inhibitors as well as more results in the accumulation of ACh in synaptic clefts [24]. The activity effective reactivators of OP nerve agent-inhibited enzymes. Com- of AChE can be restored by treatment with a reactivator from the pounds were designed by modifying the structures of N- quaternary pyridinium oxime family, such as 2-PAM (KI for mAChE of substituted 2-hydroxyiminoacetamides, recently introduced non- € 150 mmol/L) [25], trimedoxime (KI for hAChE of 18 mmol/L) [26],Hlo- charged AChE oxime reactivators [46,47], through the introduc- 7(KI for hAChE of 24 mmol/L) [26],HI-6(KI for hAChE of 20 mmol/L) tion of a phenyl ring. It was expected that the phenyl ring would [27], (Fig. 1), by cleaving the covalent bond between the help their stabilization through p-p interactions with active site catalytic serine residue and the nerve agent [28,29].HI-6isan aromatic amino acids and provide additional binding moiety apart example of a quaternary bis-pyridinium mono-oxime that binds to from PAS- moiety and the 2-hidroxyiminoacetamide group. The both the CAS and the PAS with its two positively charged hetero- working hypothesis was that the phenyl ring would interact with cyclic aromatic rings [30].Theefficacy of both AChE inhibitors and the choline binding site directing, together with PAS-binding reactivators currently used in medical treatment of AD or OP nerve moiety, the 2-hidroxyiminoacetamide group into a so-called third agent poisoning is limited because they do not cross the blood-brain binding site surrounding Ser203. Molecular modeling was used to barrier readily due to their permanent positive charge [31]. determine and visualize the binding modes of the new compounds Butyrylcholinesterase (BChE, E.C. 3.1.1.8) is related to AChE and it and their interactions with the enzymes. can also catalyze the hydrolysis of ACh; moreover, it serves as a co- regulator of cholinergic neurotransmission [32,33]. However, BChE 2. Materials and methods plays an important role in the pathogenesis of AD with its activity increased at the early stage of disease and involvement in the am- 2.1. Chemicals yloid b-peptide aggregation developing into senile plaque deposits [34,35]. The inhibition of BChE may thus be beneficial in the medical N-substituted 2-hydroxyiminoacetamides N-(3-azido-1- treatment of AD patients. AChE and BChE show a high resemblance phenylpropyl)-2-hydroxyiminoacetamide (1), N-(3-(4- with sequence homology of 65% [35,36]. However, their active sites cyclopentyl-1H-1,2,3-triazol-1-yl)-1-phenylpropyl)-2- display different amino acids composition and therefore the BChE hydroxyiminoacetamide (2), 2-hydroxy-imino-N-(3-(4-((2- active site is about 200 Å3 bigger [37,38],consequentlyallowingthe methyl-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-1- BChE to bind and hydrolyze larger ligands and substrates than AChE phenylpropyl)acetamide (3), and 2-hydroxyimino-N-(3-(4-((2- [39]. Moreover, differences between AChE and BChE active site hydroxyiminomethyl)-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol- amino acid composition lead to AChE/BChE selectivity for many li- 1-yl)-1-phenylpropyl)acetamide (4) were synthesized. 1 was pre- gands and substrates [23,40]. Some of the selective AChE inhibitors pared via BOP catalyzed amidation of hydroxyiminoacetic acid with are BW284C51, huperzin A, and the aforementioned donepezil 3-azido-1-phenylpropylamine [48]. 2e4 were prepared by copper 124 N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 catalyzed azide-alkyne cycloaddition starting from 1 and a corre- (dissociation) constant of a complex formed at the catalytic site, aKI sponding alkyne: ethynylcyclopentane, 2-methyl-1-(prop-2-yn-1- is the Michaelis complexeoxime inhibition (dissociation) constant yl)-1H-imidazole, and 1-(prop-2-yn-1-yl)-1H-imidazole-2- of a complex formed at the peripheral site, Km is a dissociation carbaldehyde oxime, respectively [49,50]. For more detailed infor- constant of the Michaelis complex, and Vm is maximal activity. mation about the synthesis of 1e4, please refer to the For reactivation assay the undiluted ChE was incubated with Supplementary material. Cyclosarin [cyclohexyl methylphoshono- nerve agents cyclosarin, sarin and VX until inhibition exceeds 95%. ® fluoridate], sarin [propan-2-yl methylphosphonofluoridate] and VX Excess nerve agent was removed by column filtration using Strata [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phos- C18-E tubes (Phenomenex, USA) and the inhibition mixture was 4 phinate] were purchased from NC Laboratory, Spiez, Switzerland. times diluted with 0.1 mol/L sodium phosphate buffer (pH 7.4) containing 0.1 mmol/L oxime (1e4) to start the reactivation. After a 2.2. In vitro enzyme activity assays given time of reactivation, aliquots were taken out to measure the enzyme activity until end point was reached (24 h). The same Reversible inhibition of AChE/BChE by N-substituted 2- procedure was applied to the control containing the uninhibited hydroxyiminoacetamides was evaluated by determining the enzyme and oxime. Activities of both the control and reactivation decrease of enzyme activity in the presence of N-substituted 2- mixture were corrected for oxime-induced hydrolysis of substrate hydroxyiminoacetamides and substrate acetylthiocholine (ATCh). [52,53]. No spontaneous reactivation of the phosphorylated Enzyme activity was measured spectrophotometrically using the enzyme took place. Ellman assay with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and substrate acetylthiocholine (ATCh, 0.1e0.3 mmol/L) [51]. Horse serum BChE was purchased from Sigma Chemical Co., USA. Final BChE dilution was 600 fold. Human recombinant AChE was prepared at the Jean-Pierre Ebel Institute of Structural Biology (IBS)eDYNA- MOP, Grenoble, Rhone-Alpes,^ France and was a gift from Dr. Florian 2.3. Molecular modeling Nachon. Stock solution of human AChE was stabilized with addition of 1% BSA. ATCh and DTNB were purchased from Sigma Chemical Co., Compounds to be docked in the active site of human AChE and USA. N-substituted 2-hydroxyiminoacetamides were dissolved in human BChE were created and minimized using the MMFF94 force fi DMSO. ATCh and DTNB were dissolved in 0.1 mol/L sodium phos- eld implemented in ChemBio3D Ultra 12.0 (PerkinElmer, Inc., phate buffer (pH 7.4). The reaction mixture contained the enzyme USA). ’ suspended in 0.1 mol/L sodium phosphate buffer (pH 7.4), 0.3 mmol/ Accelrys Discovery Studio s Dock Ligands protocol (CDOCKER) fi LDTNB,N-substituted 2-hydroxyiminoacetamide and ATCh was used for the docking study with CHARMm force eld (Accelrys, (0.1e0.8 mmol/L). The final assay volume was 300 mLandthe USA) [54,55]. The crystal structure of human AChE (PDB: 1B41, enzymatic reaction was followed during 240 s at a temperature of 4PQE) [56] or human BChE (PDB: 2PM8) [57] was used as the rigid fi 25 CusingaTecanInfinite M200PRO plate reader (Tecan Group Ltd., receptor. The binding site within the AChE or BChE was de ned as Switzerland). To limit the influence of DMSO on the degree of the largest cavity in the enzyme structure surrounded by a sphere ¼ enzyme inhibition, the final content of DMSO was held constant (r 13 Å). The following steps were included in the CDOCKER whenever it exceeded 0.05%. protocol. First, a set of 20 random ligand conformations for each The inhibition constants were evaluated by the Enzyme Kinetics test compound was generated. In the following step, 20 random fi module of Graph Pad Prism version 6.01 (GraphPad Software, Inc., orientations were kept if the energy was less than the speci ed USA). The dose response curves were fitted using the Mixed Model threshold value of 300 vdW. This process continued until either a Inhibition (Scheme 1) equation: desired number of low-energy orientations were found or the maximum number of bad orientations had been attempted. The ’ $ maximum number of bad orientations was set to 800. In the next ¼ Vm S vi step each orientation was subjected to simulated annealing mo- K’ þ S m lecular dynamics. The temperature was increased to 700 K then   cooled to 310 K. The numbers of heating and cooling phase steps 1 þ I during simulated annealing were set to 2000 and 5000, respec- KI ’ ¼ $ 1  ’ ¼ $  tively. For the simulated annealing refinement, grid extension Vm Vm Km Km I I (8.0 Å) was used. In the subsequent step, a final minimization of 1 þ a 1 þ a KI KI each refined pose of the ligand in the rigid receptor is performed using full potential. In the end, for each final pose, the CHARMm where S is the concentration of substrate ATCh, I is the concen- energy (interaction energy plus ligand strain) and the interaction e tration of inhibitor (oxime), KI is the enzyme oxime inhibition energy alone are calculated. The poses are sorted by CHARMm energy and the 20 top scored (most negative, thus favorable for binding) poses are retained. The selected poses for each enzymeeligand complex were minimized using protocol Minimization with Smart Minimizer al- gorithm. The applied algorithm performs 1000 steps of Steepest Decent with a RMS gradient tolerance of 3, followed by Conjugate Gradient minimization with the values of Max Steps and RMS Gradient set to 500 and 0.01, respectively. Generalized Born with Molecular Volume implicit solvent model was used [58,59]. The non-polar surface area was used to approximate the non-polar component of the solvation energy. Implicit solvent dielectric constant was set to 80. Distance cutoff value used for counting non- Scheme 1. Reaction scheme for mixed inhibition model. bonded interaction pairs was set to 14.0 Å. N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 125

Fig. 2. Chemical structures of synthesized N-substituted 2-hydroxyiminoacetamides.

3. Results and discussion concentration range which may explain its exceptional behavior. For BChE, this decrease in ES affinity is even more pronounced 3.1. Design of compounds indicating obstruction of interactions between the oxime and gorge residues due to the substrate presence. Inhibition con- Based on the hypothesis that the dual-binding mode of inter- stants (KI) for AChE ranged from 50 to 1200 mmol/L (Table 1)with action between AChE/BChE and its inhibitors or reactivators can the inhibition potency increasing in the following order: be extended to a so-called triple-binding mode of interaction, 1 < 2 < 4 < 3. For BChE, KI ranged from 0.3 to 130 mmol/L with we designed and synthesized four new compounds capable of the same order of inhibition potency as the one observed for simultaneous non-covalent triple-binding with enzymes. In doing AChE. Results show that our modification of the presumed so, we modified the structures of recently reported N-substituted 2- PAS-binding moieties can influence inhibition potency signifi- hidroxyimnoacetamides [46] by introducing the phenyl ring. Some cantly. Compound 1 displayed the lowest affinity toward the of the reported N-substituted 2-hidroxyiminoacetamides are enzymes, which is probably the result of the lack of a more known to possess high reactivation potential toward sarin-, cyclo- elaborated structure of its presumed PAS-binding moiety, i.e. an sarin-, and VX-inhibited AChE [47]. According to our hypothesis, azide group present in 2, 3 and 4. On the other hand, 3 proved to the phenyl ring was expected to bind in the choline binding site. be the most potent inhibitor of both enzymes. Also, all four ox- The presumed PAS-binding moieties ranged from an azide group to imes demonstrated a preference for binding to BChE, probably functionalized heterocycles and were connected with the central due to a bigger BChE active site compared to AChE allowing such N-(1-phenylpropyl)-2-hydroxyiminoacetamide scaffold via a 1,2,3- bulkier ligands to adopt more favorable binding conformation triazole ring. Following N-substituted 2-hydroxyiminoacetamides [39]. Also, 6 out of 14 aromatic amino acids in the AChE active were synthesized: N-(3-azido-1-phenylpropyl)-2-hydroxyiminoa- site corresponding to aliphatic ones in the BChE site made it cetamide (1), N-(3-(4-cyclopentyl-1H-1,2,3-triazol-1-yl)-1-pheny- more hydrophobic and favorable for lipophilic compounds. lpropyl)-2-hydroxyiminoacetamide (2), 2-hydroxyimino-N-(3-(4- Moreover, 3 displayed an almost 150 times higher affinity for ((2-methyl-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-1- BChE compared to AChE, thus 3 can be considered a selective phenylpropyl)acetamide (3), and 2-hydroxyimino-N-(3-(4-((2- BChE inhibitor. hydroxyiminomethyl)-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol- 1-yl)-1-phenylpropyl)acet-amide (4)(Fig. 2).

3.2. Kinetic measurements - inhibition

To determine the binding affinity of AChE and BChE for 1e4, we performed detailed enzyme kinetics measurements (Fig. 3). All four of the N-substituted 2-hydroxyiminoacetamides reversibly inhibited both AChE and BChE displaying mixed types of inhibition. This suggests that all four oximes can bind to the free enzyme (E) and to the Michaelis complex (ES). Parameter a > 1 describes the decrease in the ES complex affinity for an oxime in comparison to the affinity of the free enzyme (E). All of the tested oximes bind more weakly to the ES Fig. 3. Representative plot of AChE activity and the effect of substrate concentration on than to the E in the case of AChE, except for 2. However, due to AChE activity in the presence and absence of N-substituted 2-hydroxyiminoacetamide the low solubility of 2 in 0.1 M sodium phosphate buffer, its ki- 3. To limit the influence of DMSO on the degree of enzyme inhibition the final content netic parameters could not be evaluated at the optimal of DMSO was held constant at 0.1%. 126 N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132

Table 1 Reversible inhibition of human recombinant acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by N-substituted 2-hydroxyiminoacetamides 1e4.

N-substituted 2-hydroxyiminoacetamide [ATCh]/mmol/L [N-substituted 2- hydroxyiminoacetamide]/mmol/L KI/mmol/L a AChE 1 0.1e0.6 500e1500 1187 ± 248 2.5 ± 1.0 2 0.1e0.6 30e150a 358 ± 197 0.9 ± 0.7 3 0.1e0.8 15e80 49 ± 22 2.9 ± 1.5 4 0.1e0.8 50e200 135 ± 26 4.8 ± 1.3 BChE 1 0.1e0.8 50e200 132 ± 14 6.3 ± 2.2 2 0.1e0.8 15e75 42 ± 415± 10 3 0.1e0.8 0.1e0.4 0.33 ± 0.03 6.0 ± 1.7 4 0.1e0.8 20e100 30 ± 3 5.7 ± 1.8

a Due to the low solubility of 2 in 0.1 M sodium phosphate buffer, pH 7.4, the constant could not be evaluated at higher concentration of 2.

3.3. Kinetic measurements - reactivation

Compounds 1e4 were screened for reactivation of cyclosarin-, sarin- and VX-inhibited AChE and BChE. Their reactivation effi- cacy was presented as maximal reactivation after a given period of time (Fig. 4). Maximal reactivation ranged from 3% for 1 in the case of sarin-inhibited AChE to 78% to 4 inthecaseofcyclosarin- inhibited AChE. Compound 1 showed a better reactivation effi- cacy in the case of sarin-inhibited BChE with a maximal reac- tivation of 51% compared to sarin-inhibited AChE. The highest reactivation overall efficacy was for 2 with a maximal reac- tivation of 85% in the case of VX-inhibited BChE. Generally, compounds 1e4 were better reactivators of - inhibited BChE. It is interesting to note that compound 4 is a bis-oxime; its highest efficacy in the reactivation of organophosphate-inhibited AChE may be explained by the increased number of oxime groups compared to the other tested compounds. Surprisingly, this was not observed in the case of organophosphate-inhibited BChE meaning that interactions of 4 with BChE gorge residues somewhat reduced reactivation effi- cacy. Finding where bis-oximes had a smaller expected higher reactivation efficacy than their mono-oxime analogues, where the second oxime group in the bis-oxime molecule is replaced with an amide group, describes a complicated nature of ChE reactivation process [60].

3.4. Molecular modeling

In order to reveal the key interactions leading to the observed differences in binding affinity of 1e4 and their preference for binding to BChE, molecular docking studies were conducted using structures of human AChE (PDB: 1B41, 4PQE) and human BChE (PDB: 2PM8) (Figs. 5 and 6). 1e4 were docked into the active site of the enzyme. The resulting poses were critically investigated targeting the ones including p-p interactions between a com- pound and aromatic amino acids of PAS and choline binding sites e a type of interaction typical of enzyme-inhibitor/reactivator complex observed by X-ray crystallography for both AChE and Fig. 4. End point profile for reactivation of cyclosarin-, sarin- and VX-inhibited cho- fi e BChE [12,18e23,30,61,62]. Poses that had fulfilled these criteria linesterases. Ef cacy of oxime compounds 1 4 (0.1 mmol/L) are shown in reactivation of cyclosarin-, sarin- and VX-inhibited AChE (A) and BChE (B). Reactivation was were chosen for the prediction of key interactions between the monitored up to 24 h. compound and the enzyme summarized in Table 2. In agreement with experimentally determined enzymeeN-substituted 2- hydroxyiminoacetamide inhibition constants, 3 displayed the 3.4.1. Modeling of AChE complexes highest number of predicted interactions with both enzymes, The predicted binding geometry of 1 in the AChE active site while the weakest inhibitor 1 displayed the lowest number of supported our hypothesis that a phenyl ring would bind in the interactions. On the other, it was clear that the differences in in- choline binding site yielding p-p interactions with Trp86 (Fig. 5A). hibition potencies and their preference for binding to BChE could As mentioned, this type of interaction has been confirmed from not be attributed solely to the number of interactions with the structures for numerous complexes of ChEs and aromatic ring- enzyme. containing ligands, i.e. parallel pep stacking between the benzyl N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 127

Table 2 List of N-substituted 2-hydroxyiminoacetamideeenzyme interactions.

Enzyme N-substituted 2-hydroxyiminoacetamide Interactionsa

AChE 1 D) Tyr133 A) Ser203, His447 p-p) Trp86 2 D) Tyr124, Ser203, Tyr337 A) none p-p) Trp86 3 D) none A) Glu202 p-p) Tyr124, Trp286, Phe297 (p-sigma), Tyr341 4 D) Ser203, Phe295 A) Glu202 p-p) Trp86, Tyr341 (p-sigma) BChE 1 D) Ser198, A) Ser198, p-p) Trp82, 2 D) Ser198 A) Leu286 p-p) Trp82, Phe329 3 D) Gly116, Gly117 A) Ser198, Pro285 p-p) Trp82, His438 4 D) Thr120 A) Trp82 p-p) Trp82, Tyr332

a D) H-bond donor; A) H-bond acceptor; p-p interactions. ring of the donepezil complexed with Torpedo californica (Tc) AChE PAS where second pyridinium ring of oximes HI-6, Ortho-7, and Trp84 or human (h) AChE Trp86 [17,63], pep stacking of the tacrine obidoxime forms a p-p sandwich with Tyr124 and Trp286, and for ring against the Trp84 in the tacrineeTcAChE complex [64] and this Trp286 needs to change its conformation from one that is bistacrineeTcAChE complex [13], and against the Trp82 in the observed in apo state of AChE [30,65]. tacrineehBChE complex [61]. Also in accordance with our hy- Additionally, the 2-hydroxyiminoacetamide moiety of 3 creates pothesis, the 2-hydroxyiminoacetamide moiety was directed to- a hydrogen bond with Glu202. ward the catalytic serine Ser203 making hydrogen bonds between The predicted binding geometry of 4 in the AChE (Fig. 5D) its hydroxyl group and Ser203 and/or His447 of the catalytic triad. suggests that 4 also binds with its presumed PAS-binding moiety, Additionally, a side chain of 1 with an azide group makes hydrogen imidazole-2-carboxaldehyde oxime, at the entry of the gorge and bonds with Tyr133. This residue makes hydrogen bonds with li- the phenyl ring at the choline binding site. The triazole ring binds in gands, i.e. ()-huperzine A in complex with hAChE [61]. The lack of the narrow part defined by Tyr124 and Tyr337. The only difference interactions between 1 and the residues in the PAS region could between the predicted binding geometry of 3 and 4 was that 4 had explain its low inhibition potential when compared to other N- been buried deeper in the AChE active site. This results in p-p in- substituted 2-hydroxyiminoacetamides. teractions between the imidazole-2-carboxaldehyde oxime and Model complex of 2 and AChE (Fig. 5B) also predicts p-p in- Trp341. Additionally, 2-hydroxyiminoacetamide group makes teractions between the phenyl ring and Trp86. The triazole ring, hydrogen bonds with Glu202 and Ser203. However, this also leads absent in 1, makes hydrogen bonds with Tyr124 which is similar to to the loss of interaction between the imidazole ring and PAS res- a hydrogen bond between the phenol ring of Tyr124 and the pyr- idues Tyr124 and Trp286 which could explain the lower inhibition idinium ring of the 2-hydroxy-iminomethylpyridinium ring of HI-6 potential of 4 when compared to 3. in complex with mAChE [65]. These interactions seem to govern the overall binding mode of 2 in the AChE active site directing the 3.4.2. Modeling of BChE complexes presumed PAS-binding moiety, i.e. the cyclopentyl ring, in the PAS The most commonly observed change in the predicted binding and the 2-hydroxyiminoacetamide moiety toward the catalytic geometry of 1e4 in the BChE active site when compared to those for serine with which it makes hydrogen bonds via its oxime group AChE has to do with the energetically more favorable bended [62]. conformations of oximes in the BChE active site, a finding which Elongated binding conformation of 3 in the AChE active site reflects the larger BChE active site volume. The complex of 1 in the (Fig. 5C) is characterized with a methylimidazole ring, directed BChE active site (Fig. 6A) again supports our hypothesis that a towards the entry of the AChE gorge and the 2- phenyl ring would bind in the choline binding site making p-p hydroxyiminoacetamide moiety directed towards the bottom of interactions with Trp82. However, in the BChE active site the the gorge. The geometry of 3 in the AChE gorge leads to multiple p- phenyl ring is placed closer to the center of the choline binding site. p interactions with aromatic amino acids; the imidazole ring with This change in the positioning of the phenyl ring is made possible Trp286, the triazole ring with Tyr341 and with Phe297 (p-sigma because bulky Tyr337 in the AChE corresponds to smaller Ala328 in interaction), and the phenyl ring interacts with Tyr124. This is in the BChE. Additionally, the side chain modified with an azide group accordance with X-ray structures of oximes HI-6, Ortho-7, and is oriented almost parallel to the Trp82 main chain making pep obidoxime in complex with mAChE [65], and donepezil in complex interactions with its indole ring and the 2-hydroxyiminoacetamide with TcAChE [17]. There is also an alternative binding of oximes in moiety is directed towards Ser203 making multiple hydrogen Fig. 5. The stereo view of docked conformation of the N-substituted 2-hydroxyiminoacetamides 1e4 (AeD, respectively) in the active site of the AChE. Non-covalent interactions are shown as green dashed lines (H-bonds) and orange lines (p interactions). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 6. The stereo view of docked conformation of the N-substituted 2-hydroxyiminoacetamides 1e4 (AeD) in the active site of the BChE. Non-covalent interactions are shown as green dashed lines (H-bonds) and orange lines (p interactions). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 130 N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 bonds. Once again, the lack of interactions between 1 and the It can be concluded from molecular docking studies that the residues in the PAS region could explain its lowest inhibition po- predicted binding modes of 2e4 in the AChE active site support tential among all of the tested compounds. our hypothesis of a so-called triple-binding mode of interaction The complex of 2 and BChE (Fig. 6B) predicts binding for 2 with the enzyme, with the presumed PAS-binding moiety in the almost perpendicularly to the axis connecting the entry and the PAS, the phenyl ring in the choline binding site, and the 2- bottom of the active site gorge. This orientation is made possible hydroxyiminoacetamide moiety in the third binding site sur- because aromatic Tyr124, Phe297, and Tyr337 in the AChE active rounding active serine. On the other hand, none of the N- site correspond to smaller Gln119, Val288, and Ala328 in the BChE substituted 2-hydroxyiminoacetamides is predicted to bind in active site, respectively. Otherwise, these aromatic amino acids in the BChE active site in complete accordance with the above the AChE active site would not allow such an orientation of 2. stated hypothesis, though all three binding groups are involved in Thereby, the cyclopentyl ring occupies the space normally inac- non-covalent interactions with the enzyme. However, most of cessible in the AChE active site. 2-hydroxyiminoacetamide moiety the predicted interactions between N-substituted 2- is directed towards the acyl pocket with its hydroxyl group and hydroxyiminoacetamides and ChEs could be supported with in- making hydrogen bonds with Leu286. Additionally, the carbonyl teractions observed from the X-ray structures of various ligands oxygen of the 2-hydroxyiminoacetamide moiety makes hydrogen complexed with ChE. To the best of our knowledge, only a few bonds with Ser198. Contrary to our hypothesis, the phenyl ring is interactions in the BChE active site, namely p-p interactions be- shifted towards the entry of the BChE active site gorge, but is just tween His438 and the methylimidazole ring of 3, hydrogen bond close enough to Phe329 to make a p-p interaction. This distinct between Phe295 and the 2-hydroxyiminoacetamide moiety of 3, residue corresponds to Phe338 in the AChE active site which par- hydrogen bond between Trp82 and the 2-hydroxyiminoacetamide ticipates in aromatic interactions, the donepezil complex with moiety of 4, and the hydrogen bond between Thr120 and the 2- TcAChE [17] and the HI-6 complex with mAChE [65]. hydroxyiminoacetamide moiety of 4 could not have been sup- Model complex of 3 and BChE (Fig. 6C) predicts geometry where ported with interactions revealed by X-ray crystallography. The 3 is in a bended conformation and is placed in the center of the differences in the predicted binding modes of 1e4 between the BChE active site. Once again this is expected due to Gln119, Val288, two enzymes reflect different stereoelectronic properties of their and Ala328. Thereby, the methylimidazole ring occupies the vol- active sites caused because 6 out of 14 aromatic amino acids in the ume otherwise restricted in the AChE active site because of the AChE active site corresponded to aliphatic ones in the BChE active Tyr337 side chain and interacts with Trp82 and His438 via p-p site. The most important ones that primarily governed the dif- interactions. The 2-hydroxyiminoacetamide moiety is directed to- ferences in the predicted binding modes of 1e4 occurred at the wards the bottom of the active site where its amide hydrogen position of Tyr72, Tyr124, Phe297 and Tyr337 in the AChE active makes hydrogen bonds with the backbone of Pro285 and the oxime site. group makes hydrogen bonds with Gly116 and Gly117 from an Knowledge of the degree of AChE/BChE selectivity of oxime oxyanion hole, as well as with Ser198. It has been assumed that compounds is may be important for more successful treatment in hydrogen bonding with Pro285 has been partially responsible for cases of OP nerve agent poisoning. The most notable pretreat- the inhibition differences of horse, human, and mouse BChE [43]. ment strategies include protection of the AChE catalytic serine The complex of 4 and BChE (Fig. 6D) shows that the imidazole- from phosphylating agent by ligands that bind reversibly to 2-carboxaldehyde oxime and the following triazole ring are located AChE [66e70] and the use of bioscavengers, i.e. BChE, prone to in the upper part of the active site while the phenyl ring and the 2- inhibition by a phosphylating agent [71,72]. Furthermore, BChE is hydroxyiminoacetamide moiety are located close to the bottom of considered to act as a natural bioscavenger in the bloodstream the active site. Such positioning of the triazole ring allows it to [73,74]. Considering the especially high degree of preference make p-p interactions with Tyr332 that corresponds to Tyr341 in for binding to BChE displayed by N-substituted 2- the AChE site. In support of our hypothesis, the phenyl ring hydroxyiminoacetamide 3, our results clearly discourage its use was placed at the bottom of the BChE active site close enough to in the protection of the AChE catalytic site from a phosphylating yield a p-p interaction with Trp82. This distinct tryptophan agent by reversible inhibition of AChE. Moreover, it would also residue is also involved in p-sigma interaction with a hydrogen diminish the BChE endogenous bioscavenging capability by atom from one of the 4 methylene groups and Trp82 main chain inhibiting BChE if administered prior to the OP nerve agent makes a hydrogen bond with the hydroxyl group of the 2- exposure. However, if its preference for binding to BChE proved hydroxyiminoacetamide moiety. The former interaction could be to be retained in the case of a phosphylated enzyme and was related to that between the methylene group in the tetrahy- followed by a fast reactivation, together with BChE it could make droazepine ring of ()-galanthamine in complex with TcAChE [62]. an enzyme-oxime pair acting as a pseudo catalytic-scavenger The 2-hydroxyiminoacetamide moiety is located close to the center [75]. Screening showed higher efficacy of compounds 1e4 in of the BChE active site and is almost perpendicular to the axis reactivation of cyclosarin-, sarin- and VX-inhibited BChE connecting the entry and the bottom of the active site gorge. Also, it comparing to AChE which supports possible bioscavenging is directed into the area between the Trp82 main chain and Thr120 capability of tested compounds. side chain with which it makes another hydrogen bond via its carbonyl oxygen. Conflict of interest 4. Conclusion The authors declare that there are no conflicts of interest asso- ciated with this work. All four compounds reversibly inhibited BChE with inhibition constants ranging from 0.30 mmol/L to 130 mmol/L. The inhibition potency of compounds increased in the following order: Acknowledgements 1 < 2<4 < 3. AChE was also reversibly inhibited by all compounds with the same order of inhibition potency. Inhibition constants The authors thank Dr. Florian Nachon for recombinant human ranged from 50 mmol/L to 1200 mmol/L. All of the compounds dis- AChE. This study was supported by the Croatian Science Foundation played a higher preference for binding to BChE. (Grant 4307). N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 131

Transparency document [23] Z. Radic, N.A. Pickering, D.C. Vellom, S. Camp, P. Taylor, Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyr- ylcholinesterase inhibitors, Biochemistry 32 (1993) 12074e12084. Transparency document related to this article can be found [24] S.M. Somani, J.A. Romano Jr., Chemical Warfare Agents: Toxicity at Low Levels, online at http://dx.doi.org/10.1016/j.cbi.2016.05.035. CRC Press LLC, Boca Raton, Florida, 2001. [25] Z. Kovarik, N. Ciban, Z. Radic, V. Simeon-Rudolf, P. Taylor, Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP, Biochem. Appendix A. Supplementary data Biophys. Res. Commun. 342 (2006) 973e978. [26] Z. Kovarik, M. Calic, A. Bosak, G. Sinko, D. Jelic, In vitro evaluation of aldoxime interactions with human acetylcholinesterase, Croat. Chem. Acta 81 (2008) Supplementary data related to this article can be found at http:// 47e57. dx.doi.org/10.1016/j.cbi.2016.05.035. [27] G. Sinko, J. Brglez, Z. Kovarik, Interactions of pyridinium oximes with acetylcholinesterase, Chem. Biol. Interact. 187 (2010) 172e176. [28] M.P. Stojiljkovic, M. Jokanovic, Pyridinium oximes: rationale for their selec- References tion as causal antidotes against organophosphate poisonings and current solutions for auto-injectors, Arh. Hig. Rada. Toksikol. 57 (2006) 435e443. [1] J.L. Taylor, R.T. Mayer, C.M. Himel, Conformers of acetylcholinesterase - a [29] F. Worek, P. Eyer, N. Aurbek, L. Szinicz, H. Thiermann, Recent advances in mechanism of allosteric control, Mol. Pharm. 45 (1994) 74e83. evaluation of oxime efficacy in nerve agent poisoning by in vitro analysis, [2] E.K. Perry, B.E. Tomlinson, G. Blessed, K. Bergmann, P.H. Gibson, R.H. Perry, Toxicol. Appl. Pharmacol. 219 (2007) 226e234. € € € Correlation of cholinergic abnormalities with senile plaques and mental test [30] F. Ekstrom, A. Hornberg, E. Artursson, L.G. Hammarstrom, G. Schneider, scores in senile dementia, Br. Med. J. 2 (1978) 1457e1459. Y.P. Pang, Structure of HI-6-sarin-acetylcholinesterase determined by X-ray [3] A. Enz, R. Amstutz, H. Boddeke, G. Gmelin, J. Malonowski, Brain selective in- crystallography and molecular dynamics simulation: reactivator mechanism hibition of acetylcholinesterase: a novel approach to therapy for Alzheimer’s and design, PLoS One 4 (2009) e5957, http://dx.doi.org/10.1371/ disease, Prog. Brain Res. 98 (1993) 431e435. journal.pone.0005957. [4] P. Taylor, Z. Radic, The cholinesterases: from genes to proteins, Annu. Rev. [31] P. Taylor, Anticholinesterase agents, in: L.L. Brunton, B.A. Chabner, Pharmacol. Toxicol. 34 (1994) 281e320. B.C. Knollman (Eds.), Goodman and Gilman’s the Pharmacological Basis of [5] A. Ordentlich, D. Barak, C. Kronman, Y. Flashner, M. Leitner, Y. Segall, N. Ariel, Therapeutics, McGraw-Hill, New York, 2011, pp. 239e254. S. Cohen, B. Velan, A. Shafferman, Dissection of the human - [32] V. Kumar, Introduction to cholinesterase inhibitors used in Alzheimer’s dis- terase active centre determinants of substrate specificity. Identification of ease therapy, in: R. Becker, E. Giacobini (Eds.), Alzheimer Disease: Therepeutic residues constituting the anionic site, the hydrophobic site, and the acyl Strategies, Birkauser,€ Boston, 1994, pp. 99e102. pocket, J. Biol. Chem. 268 (1993) 17083e17095. [33] M.M. Mesulam, A. Guillozet, P. Shaw, A. Levey, E.G. Duysen, O. Lockridge, [6] M.F. Eskander, N.G. Nagykery, E.Y. Leung, B. Khelghati, C. Geula, Acetylcholinesterase knockouts establish central cholinergic pathways and is a potent inhibitor of acetyl- and butyrylcholinesterase in Alzheimer’s pla- can use butyrylcholinesterase to hydrolyze acetylcholine, Neuroscience 110 ques and tangles, Brain Res. 1060 (2005) 144e152. (2002) 627e639. [7] C. Galdeano, E. Viayna, P. Arroyo, A. Bidon-Chanal, J.R. Blas, D. Munoz-Torrero, [34] S. Darvesh, D.A. Hopkins, C. Geula, Nat. Neurobiology of butyrylcholinesterase, F.J. Luque, Structural determinants of the multifunctional profile of dual Neurosci 4 (2003) 131e138. binding site acetylcholinesterase inhibitors as anti-Alzheimer agents, Curr. [35] V.N. Talesa, Acetylcholinesterase in Alzheimer’s disease, Mech. Ageing Dev. Pharm. Des. 16 (2010) 2818e2836. 122 (2001) 1961e1969. [8] R.W. Zhang, X.C. Tang, Y.Y. Han, G.W. Sang, Y.D. Zhang, Y.X. Ma, C.L. Zhang, [36] Y. Nicolet, O. Lockridge, P. Masson, J.C. Fontecilla-Camps, F. Nachon, Crystal R.M. Yang, Drug-evaluation of huperzine-a in the treatment of senile memory structure of human butyrylcholinesterase and of its complexes with substrate disorders, Acta Pharmacol. Sin. 12 (1991) 250e252. and products, J. Biol. Chem. 278 (2003) 41141e41147. [9] R. Wang, H. Yan, X.C. Tang, Progress in studies of huperzine A, a natural [37] H. Dvir, I. Silman, M. Harel, T.L. Rosenberry, J.L. Sussman, Acetylcholinesterase: from Chinese herbal medicine, Acta Pharmacol. Sin. from 3D structure to function, Chem. Biol. Interact. 187 (2010) 10e22. 27 (2006) 1e26. [38] N.A. Çokugras, Butyrylcholinesterase: structure and physiological importance, [10] Z. Radic, P. Taylor, Interaction kinetics of reversible inhibitors and substrates Turk. J. Biochem. 28 (2003) 54e61. with acetylcholinesterase and its 2 complex, J. Biol. Chem. 276 [39] A. Saxena, A.M.G. Redman, X. Jiang, O. Lockridge, B.P. Doctor, Differences in (2001) 4622e4633. active-site gorge dimensions of cholinesterase revealed by binding of in- [11] Y. Bourne, P. Taylor, Z. Radic, P. Marchot, Structural insights into ligand in- hibitors to human butyrylcholinesterase, Chem. Biol. Interact. 119120 teractions at the acetylcholinesterase peripheral anionic site, Embo J. 22 (1999) 61e69. (2003) 1e12. [40] P. Taylor, Z. Radic, N.A. Hosea, S. Camp, P. Marchot, H.A. Berman, Structural [12] P.R. Carlier, Y.F. Han, E.S.H. Chow, C.P.L. Li, H. Wang, T.X. Lieu, H.S. Wong, bases for the specificity of cholinesterase catalysis and inhibition, Toxicol. Lett. Y.P. Pang, Evaluation of short-tether bis-THA AChE inhibitors. A further test of 8283 (1995) 453e458. the dual binding site hypothesis, Bioorg. Med. Chem. 7 (1999) 351e357. [41] Z. Kovarik, Z. Radic, B. Grgas, M. Skrinjaric-Spoljar, E. Reiner, V. Simeon- [13] E.H. Rydberg, B. Brumshtein, H.M. Greenblatt, D.M. Wong, D. Shaya, Rudolf, Amino acid residues involved in the interaction of acetylcholines- L.D. Williams, P.R. Carlier, Y.P. Pang, I. Silman, J.L. Sussman, Complexes of terase and butyrylcholinesterase with the Ro 02-0683 and bam- alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: buterol, and with terbutaline, Biochim. Biophys. Acta 1433 (1999) 261e271. binding of bis(5)-tacrine produces a dramatic rearrangement in the active-site [42] Z. Kovarik, V. Simeon-Rudolf, Interaction of human butyrylcholinesterase gorge, J. Med. Chem. 49 (2006) 5491e5500. variants with bambuterol and terbutaline, J. Enzym. Inhib. Med. Chem. 19 [14] W.G. Lewis, L.G. Green, F. Grynszpan, Z. Radic, P.R. Carlier, P. Taylor, M.G. Finn, (2004) 113e117. K.B. Sharpless, Click chemistry in situ: acetylcholinesterase as a reaction [43] Z. Kovarik, A. Bosak, G. Sinko, T. Latas, Exploring active sites of cholinesterases vessel for the selective assembly of a femtomolar inhibitor from an array of by inhibition with bambuterol and haloxon, Croat. Chem. Acta 76 (2003) building blocks, Angew. Chem. Int. 41 (2002) 1053e1057. 63e67. [15] Y. Bourne, H.C. Kolb, Z. Radic, K.B. Sharpless, P. Taylor, P. Marchot, Freeze- [44] M. Golicnik, G. Sinko, V. Simeon-Rudolf, Z. Grubic, J. Stojan, Kinetic model of frame inhibitor captures acetylcholinesterase in a unique conformation, Proc. ethopropazine interaction with horse serum butyrylcholinesterase and its Natl. Acad. Sci. U. S. A. 101 (2004) 1449e1454. docking into the active site, Arch. Biochem. Biophys. 398 (2002) 23e31. [16] A. Inoue, T. Kawai, M. Wakita, Y. Imura, H. Sugimoto, Y. Kawakami, The [45] G. Sinko, Z. Kovarik, E. Reiner, V. Simeon-Rudolf, J. Stojan, Mechanism of simulated binding of (þ/e)-2,3-dihydro-5,6-dimethoxy-2-[(1-(phenyl- stereoselective interaction between butyrylcholinesterase and ethopropazine methyl)-4-piperidinyl)methyl]-1H-inden-1-one hydrochloride (E2020) and enantiomers, Biochimie 93 (2011) 1797e1807. related inhibitors to free and acylated and corre- [46] R.K. Sit, Z. Radic, V. Gerardi, L. Zhang, E. Garcia, M. Katalinic, G. Amitai, sponding structure-activity analyses, J. Med. Chem. 39 (1996) 4460e4470. Z. Kovarik, V.V. Fokin, K.B. Sharpless, P. Taylor, New structural scaffolds for [17] G. Kryger, I. Silman, J.L. Sussman, Structure of acetylcholinesterase complexed centrally acting oxime reactivators of phosphylated cholinesterases, J. Biol. with E2020 (Aricept (R)): implications for the design of new anti-Alzheimer Chem. 286 (2011) 19422e19430. drugs, Structure 7 (1999) 297e307. [47] Z. Kovarik, N. Macek, R.K. Sit, Z. Radic, V.V. Fokin, K.B. Sharpless, P. Taylor, [18] D.A. Dougherty, D.A. Stauffer, Acetylcholine binding by a synthetic receptor: Centrally acting oximes in reactivation of tabun-phosphoramidated AChE, implications for biological recognition, Science 250 (1990) 1558e1560. Chem. Biol. Interact. 203 (2013) 77e80. [19] D.A. Dougherty, Cation-pi interactions in chemistry and biology: a new view [48] A. Knezevi c, V. Vinkovic, N. Marakovic, G. Sinko, Enzyme-catalysed cascade of benzene, Phe, Tyr, and Trp, Science 271 (1996) 163e168. synthesis of hydroxyiminoacetamides, Tetrahedron Lett. 55 (2014) [20] I. Silman, M. Harel, J. Eichler, J.L. Sussman, A. Anselmet, J. Massoulie, Structure- 4338e4341. function relationships in the binding of reversible inhibitors in the active-site [49] V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless, A stepwise huisgen gorge of acetylcholinesterase, in: R. Becker, E. Giacobini (Eds.), Alzheimer cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides Disease: Therepeutic Strategies, Birkauser,€ Boston, 1994, pp. 88e92. and terminal alkynes, Angew. Chem. Int. 41 (2002) 2596e2599. [21] Z. Radic, R. Duran, D.C. Vellom, Y. Li, C. Cervenansky,~ P. Taylor, Site of fasciculin [50] P.K. Avti, D. Maysinger, A. Kakkar, Alkyne-azide “click” chemistry in designing interaction with acetylcholinesterase, J. Biol. Chem. 269 (1994) 11233e11239. nanocarriers for applications in biology, Molecules 18 (2013) 9531e9549. [22] Y. Bourne, P. Taylor, P. Marchot, Acetylcholinesterase inhibition by fasciculin: [51] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, A new and rapid crystal structure of the complex, Cell 83 (1995) 503e512. colorimetric determination of acetylcholinesterase activity, Biochem, 132 N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132

Pharmacol 7 (1961) 88e95. residues in the active-site gorge of acetylcholinesterase, Proc. Natl. Acad. Sci. [52] G. Sinko, M. Calic, Z. Kovarik, para- and ortho-Pyridinium aldoximes in re- U. S. A. 90 (1993) 9031e9035. action with acetylthiocholine, FEBS Lett. 580 (2006) 3167e3172. [65] F. Ekstrom,€ Y.-P. Pang, M. Boman, E. Artursson, C. Akfur, S. Borjegren,€ Crystal [53] G. Sinko, M. Calic, A. Bosak, Z. Kovarik, Limitation of the Ellman method: structures of acetylcholinesterase in complex with HI-6, Ortho-7 and obi- cholinesterase activity measurement in the presence of oximes, Anal. Bio- doxime: structural basis for differences in the ability to reactivate tabun chem. 370 (2007) 223e227. conjugates, Biochem. Pharmacol. 72 (2006) 597e607. [54] B.R. Brooks, R.E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan, [66] L.W. Harris, W.C. Heyl, D.L. Stitcher, C.A. Broomfield, Effects of 1.1-oxydi- M. Karplus, CHARMM: a program for macromolecular energy, minimization, methylene bis-(4-tert-butylpyridinium chloride) (SAD-128) and decametho- and dynamics calculations, J. Comp. Chem. 4 (1983) 187e217. nium on reactivation of soman and sarin-inhibited cholinesterase by oximes, [55] F.A. Momany, R. Rone, Validation of the general purpose QUANTA 3.2/ Biochem. Pharmacol. 27 (1978) 757e761. CHARMm force field, J. Comp. Chem. 13 (1992) 888e900. [67] J. Bajgar, Prophylaxis against organophosphorus poisoning, J. Med. Che. Def. 1 [56] G. Kryger, M. Harel, K. Giles, L. Toker, B. Velan, A. Lazar, C. Kronman, D. Barak, (2004) 1e16. N. Ariel, A. Shafferman, I. Silman, J.L. Sussman, Structures of recombinant [68] E. Reiner, Inhibition of acetylcholinesterase by 4,4-bipyridine and its effect native and E202Q mutant human acetylcholinesterase complexed with the upon phosphylation of the enzyme, Croat. Chem. Acta 59 (1986) 925e931. snake-venom toxin fasciculin-II, Acta Crystallogr. Sect. D. 56 (2000) [69] G. Lallement, V. Baille, D. Baubichon, P. Carpentier, J.M. Collombet, P. Filliat, 1385e1394. A. Foquin, E. Four, C. Masqueliez, G. Testylier, L. Tonduli, F. Dorandeu, Review [57] M.N. Ngamelue, K. Homma, O. Lockridge, O.A. Asojo, Crystallization and X-ray of the value of huperzine as pretreatment of organophosphate poisoning, structure of full-length recombinant human butyrylcholinesterase, Acta Neurotoxicology 23 (2002) 1e5. Crystallogr. Sect. F. 63 (2007) 723e727. [70] S. Eckert, P. Eyer, H. Muckter, F. Worek, Kinetic analysis of the protection [58] M. Feig, C.L. Brooks III, Recent advances in the development and application of afforded by reversible inhibitors against irreversible inhibition of acetylcho- implicit solvent models in biomolecule simulations, Curr. Opin. Struct. Biol. 14 linesterase by highly toxic organophosphorus compounds, Biochem. Phar- (2004) 217e224. macol. 72 (2006) 344e357. [59] M. Nina, D. Beglov, B. Roux, Atomic Born radii for continuum electrostatic [71] M.A. Dunn, B.E. Hackley, F.R. Sidell, Pretreatment for nerve agent exposure, in: calculations based on molecular dynamics free energy simulations, J. Phys. F.R. Sidell, E.T. Takafuji, D.R. Franz (Eds.), Medical Aspects of Chemical Bio- Chem. B 101 (1997) 5239e5248. logical Warfare, Walter Reed Army Medical Center, Washington, 1997, pp. [60] Z. Kovarik, M. Calic, G. Sinko, A. Bosak, S. Berend, A.L. Vrdoljak, B. Radic, Ox- 181e196. imes: reactivators of phosphorylated acetylcholinesterase and antidotes in [72] D.E. Lenz, C.A. Broomfield, D.M. Maxwell, D.M. Cerasoli., Nerve agent bio- therapy against tabun poisoning, Chem. Biol. Interact. 175 (2008) 173e179. scavengers: protection against high- and low-dose organophosphorus expo- [61] F. Nachon, E. Carletti, C. Ronco, M. Trovaslet, Y. Nicolet, L. Jean, P.-Y. Renard, sure, in: S.M. Somani, J.A. Romano Jr. (Eds.), Chemical Warfare Agents: Crystal structures of human cholinesterases in complex with huprine W and Toxicity at Low Levels, CRS Press LLC, Boca Raton, Florida, 2001, pp. 215e243. tacrine: elements of specificity for anti-Alzheimer’s drugs targeting acetyl- [73] L. Raveh, E. Grauer, J. Grunwald, E. Cohen, Y. Ashani, The stoichiometry of and butyryl-cholinesterase, Biochem. J. 453 (2013) 393e399. protection against soman and VX toxicity in monkeys pretreated with human [62] H.M. Greenblatt, G. Kryger, T. Lewis, I. Silman, J.L. Sussman, Structure of butyrylcholinesterase, Toxicol. Appl. Pharmacol. 145 (1997) 43e53. acetylcholinesterase complexed with (e)-galanthamine at 2.3 Å resolution, [74] P. Masson, F. Nachon, C.A. Broomfield, D.E. Lenz, L. Verdier, L.M. Schopfer, FEBS Lett. 463 (1999) 321e326. O. Lockridge, A collaborative endeavor to design cholinesterase-based cata- [63] J. Cheung, M.J. Rudolph, F. Burshteyn, M.S. Cassidy, E.N. Gary, J. Love, lytic scavengers against toxic organophosphorus esters, Chem. Biol. Interact. M.C. Franklin, J.J. Height, Structures of human acetylcholinesterase in complex 175 (2008) 273e280. with pharmacologically important ligands, J. Med. Chem. 55 (2012) [75] A. Lucic Vrdoljak, M. Calic, B. Radic, S. Berend, D. Jun, K. Kuca, Z. Kovarik, 10282e10286. Pretreatment with pyridinium oximes improves antidotal therapy against [64] M. Harel, I. Schalk, L. Ehret-Sabatier, F. Bouet, M. Goeldner, C. Hirth, tabun poisoning, Toxicology 228 (2006) 41e50. P.H. Axelsen, I. Silman, J.L. Sussman, Quaternary ligand binding to aromatic